Nucleic acid molecule

ABSTRACT

The present invention is directed generally to an isolated nucleic acid molecule encompassing a neocentromere or a functional derivative thereof or a latent, synthetic or hybrid form thereof and its use inter alia in developing a range of eukaryotic artificial chromosomes including mammalian (e.g. human) and non-mammalian an artificial chromosomes. Such artificial chromosomes are useful in a range of genetic therapies.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.09/078,294, filed on May 13, 1998 now U.S. Pat. No. 6,265,211.

FIELD OF THE INVENTION

The present invention is directed generally to an isolated nucleic acidmolecule encompassing a neocentromere or a functional derivative thereofor a latent, synthetic or hybrid form thereof and its use inter alia indeveloping a range of eukaryotic artificial chromosomes includingmammalian (e.g. human) and non-mammalian artificial chromosomes. Suchartificial chromosomes are useful in a range of genetic therapies.

BACKGROUND OF THE INVENTION

Bibliographic details of the publications referred to by author in thisspecification are collected at the end of the description.

The rapidly increasing sophistication of recombinant DNA technology isgreatly facilitating research and development in the medical and alliedhealth fields. A particularly important area is in mammalian includinghuman genetics and the molecular mechanisms behind some geneticabnormalities. Progress in research in this area has been hampered bythe lack of a cloned nucleic acid molecule encompassing a humancentromere. The identification and cloning of a human centromere willpromote the development of techniques for introducing genes intoeukaryotic cells and in particular mammalian including human cells andwill be an important asset to gene therapy and the development of arange of genetic diagnostic tests.

The centromere is an essential structure for sister chromatid cohesionand proper chromosomal segregation during mitotic and meiotic celldivisions. The centromere of the budding yeast Saccharomyces cerevisiaehas been extensively studied and shown to be contained within arelatively short DNA segment of 125 bp that is organized into an 8-bp(CDEI) and 26-bp (CDEIII) domain, separated by a 78- to 87-bp, highlyAT-rich, middle (CDEII) domain (Clarke and Carbon, 1985). The centromereof the fission yeast Schizosaccharomyces pombe is considerably larger,ranging from 40 to 100 kb, and consists of a central core DNA element of4 to 7 kb flanked on both sides by inverted repeat units (Steiner etal., 1993). Recently, the functional DNA components of a highereukaryotic centromere have been characterized in a minichromosome fromDrosophila melanogaster and shown to consist of a 220-kb essential coreDNA flanked by 200 kb of highly repeated sequences on one side (Murphyand Karpen, 1995).

The mammalian centromere, like the centromeres of all higher eukaryotesstudied to date, contains a great abundance of highly repetitive,heterochromatic DNA. For example, a typical human centromere contains 2to 4 Mb of the 171-bp α-satellite repeat (Wevrick and Willard 1989,1991; Trowell et al., 1993), plus a smaller and more variable quantityof a 5-bp satellite III DNA (Grady et al., 1992; Trowell et al., 1993).The role of these satellite sequences is presently unclear. Transfectionof a cloned 17-kb uninterrupted α-satellite array into cultured simiancells (Haaf et al., 1992) or a 120-kb α-satellite-containing YAC intohuman and hamster cells (Larin et al., 1994) appear to confer centromerefunction at the sites of integration. Other workers have analyzedrearranged Y chromosomes (Tyler-Smith et al., 1993), or dissected thecentromere of the human Y chromosome with cloned telomeric DNA (Brown etal., 1994) and suggested that 150 to 200 kb of α-satellite DNA plus ˜300kb of adjacent sequences are associated with human centromere function.In addition, a human X-derived minichromosome that retained 2.5 Mb ofα-satellite array has been produced by telomere-associated chromosomefragmentation (Farr et al., 1995). In all these studies, it is not knownwhether non-α-satellite DNA sequences are embedded within thecentromeric site and operate independently of, or in concert with, theα-satellite DNA.

In mammals, four constitutive centromere-binding proteins, CENP-A,CENP-B, CENP-C, and CENP-D, have been characterized to varying extentsand implicated to have possible direct roles in centromere function.CENP-A, a protein localized to the outer kinetochore domain, is acentromere-specific core histone that shows sequence homology to thehistone H3 protein and may serve to differentiate the centromere fromthe rest of the chromosome at the most fundamental level of chromatinstructure—the nucleosome (Sullivan et al., 1994). CENP-B, a proteinwhich associates with the centromeric heterochromatin through itsbinding to the CENP-B box motif found in primate α-satellite and mouseminor satellite DNA, probably has a role in packaging centromericheterochromatic DNA—a role which, however, may not be indispensablesince the protein is undetectable on the Y chromosome (Pluta et al.,1990) and is found on the inactive centromeres of dicentric chromosomes(Earnshaw et al., 1989). CENP-C has been shown to be located at theinner kinetochore plate and is postulated to have an essential althoughyet undetermined centromere function, as seen, for example, frominhibition of mitotic progression following microinjection ofanti-CENP-C antibodies into cells (Bernat et al., 1990; Tomkiel et al.,1994) and from its association with the active but not the inactivecentromeres of dicentric chromosomes (Earnshaw et al., 1989; Page etal., 1995; Sullivan and Schwartz, 1995). Finally, CENP-D (or RCC1) is aguanine exchange factor that appears to have a general cellular rolethat is neither specific nor clear for the centromere (Kingwell andRattner 1987; Bischoff et al., 1990; Dasso, 1993). More recently, a newrole for the mammalian centromere as a “marshalling station” for a hostof “passenger proteins” (such as INCENPs, MCAK, CENP-E, CENP-F, 3F3/2antigens, and cytoplasmic dynein), has been recognized (reviewed byEarnshaw and Mackay, 1994, and Pluta et al., 1995). These passengerproteins, whose appearance at the centromere is transient and tightlyregulated by the cell cycle, provide vital functions that include motormovement of chromosomes, modulation of spindle dynamics, nuclearorganization, intercellular bridge structure and function, sisterchromatid cohesion and release, and cytokinesis. At present, except forCENP-B, none of the constitutive or passenger proteins have beendemonstrated to bind mammalian centromere DNA directly.

In work leading up to the present invention, the inventors identified ina patient (hereinafter referred to as “BE”) an unusual human markerchromosome, mardel 10, which is 100% stable in mitotic division both inpatient BE and in established fibroblast and transformed lymphoblastcultures. In accordance with the present invention, a region of themardel (10) chromosome has been cloned together with the correspondingregion from a normal human subject. The nucleic acid molecules clonedcontain no substantial α-satellite repeats yet are mitotically stable.The nucleic acid molecules encompass therefore, a new form of centromerereferred to herein as a “neocentromere”. The identification and cloningof a eukaryotic neocentromere without substantial α-satellite DNA repeatsequences now provides the means of generating a range of eukaryoticartificial chromosomes such as mammalian including human artificialchromosomes with uses in genetic therapy, transgenic plant and animalproduction and recombinant protein production. A range of diagnosticreagents is now also obtainable using the cloned neocentromere.

SUMMARY OF THE INVENTION

Sequence Identity Numbers (SEQ ID NOs.) for the nucleotide sequencesreferred to in the specification are defined following the bibliography.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, or variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated integer or group of integers but not the exclusion of anyother integer or group of integers.

A fibroblast cell line 920158 carrying the mardel marker chromosome wasdeposited at the European Collection of Cell Cultures (ECACC), Centrefor Applied Microbiology Research, Salisbury, Wiltshire, SP4 0JG, UK on1, May 1997 under Accession No. 97051716. Bacterial artificialchromosomes (BACs) carrying portions of the mardel (10) chromosome havealso been deposited at ECACC as follows:

-   BAC/E8-1: deposited on 5, May 1998 under Accession Number 980505016;-   BAC/F2-14: deposited on 5, May 1998 under Accession Number    980505017.

A number of human fibrosarcoma cell lines carrying variousneocentromeric constructs were deposited at ECACC as described hereafterby Accession Number with the date of deposit in parenthesises.

HT-38 98050704 (May 7, 1998) HT-47 98050705 (May 7, 1998) HT-54 98050706(May 7, 1998) HT-190 98050707 (May 7, 1998) HT-191 98050708 (May 7,1998).

One aspect of the present invention provides an isolated nucleic acidmolecule comprising a sequence of nucleotides derived from a eukaryoticchromosome and encompassing a neocentromere or a functional derivativesynthetic or hybrid form thereof which nucleic acid molecule or itsderivatives, synthetic forms or hybrid forms when introduced into acompatible cell is capable of replicating, acting as anextra-chromosomal element and segregating with cell division.

Another aspect of the present invention contemplates a nucleic acidmolecule or its chemical equivalent having a tertiary structure whichdefines a human neocentromere or a functional derivative thereof or alatent, synthetic or hybrid form thereof or its mammalian ornon-mammalian homologue.

Yet a further aspect of the present invention is directed to an isolatednucleic acid molecule comprising a sequence of nucleotides encompassinga neocentromere derived from a eukaryotic chromosome, which nucleic acidmolecule when introduced into a compatible cell is a replicating,extra-chromosomal element which segregates with cell division.

Still another aspect of the present invention is directed to an isolatednucleic acid molecule having a sequence of nucleotides or their chemicalequivalents which directs a conformation defining a human neocentromereor a functional derivative thereof or a latent, synthetic or hybrid formthereof or a mammalian or non-mammalian homologue thereof wherein theneocentromere associates with centromere binding proteins (CENP)-A andCENP-C or antibodies thereto and does not contain substantialα-satellite DNA repeat sequences.

A further aspect of the present invention is directed to an isolatednucleic acid molecule comprising a nucleotide sequence encompassing aneocentromere or a functional derivative, synthetic or hybrid formthereof which when said nucleic acid molecule is in linear form andco-introduced into a cell together with a telomeric sequence, is capableof replicating, remaining as an extra-chromosomal element and segregateswith cell division.

Another aspect of the present invention provides an isolated nucleicacid molecule or a derivative, synthetic or hybrid form thereofcomprising a sequence of nucleotides:

-   -   (i) which directs conformation defining a human neocentromere or        a functional derivative thereof or a latent, synthetic or hybrid        form thereof or its mammalian or non-mammalian homologue wherein        said neocentromere is capable of associating with CENP-A and        CENP-C;    -   (ii) which contains no substantial α-satellite DNA sequence        repeat; and    -   (iii) which is capable, when introduced into compatible cells,        of replication, remaining extra-chromosomal and segregating with        cell division.

Even yet another aspect of the present invention is directed to agenetic construct comprising an origin of replication for a eukaryoticcell and a nucleic acid molecule encompassing a human neocentromere or afunctional derivative thereof or a latent, synthetic or hybrid formthereof or its mammalian or non-mammalian homologue flanked by telomericnucleotide sequences functional in the cell in which the geneticconstruct is to replicate and wherein said genetic construct whenintroduced into a cell is a replicating, extra-chromosomal element whichsegregates with cell division.

Another aspect of the present invention is directed to a geneticconstruct in the form of a eukaryotic artificial chromosome such as amammalian artificial chromosome (MAC), a human artificial chromosome(HAC) or comprising an origin of replication and a sequence ofnucleotides which:

-   -   (i) directs a conformation defining a human neocentromere or a        functional derivative thereof or a latent, synthetic or hybrid        form thereof wherein said neocentromere is capable of        associating with CENP-A and CENP-C or antibodies thereto; and    -   (ii) contains no substantial α-satellite DNA repeat sequences;        said sequence of nucleotides flanked by eukaryotic (e.g.        mammalian) telomeric sequences which are in turn flanked by        yeast telomeric sequences wherein a unique enzyme site is        located between the human and yeast telomeric nucleotide        sequences such that upon contact with said enzyme, the yeast        telomeric sequences are removed and the eukaryotic (e.g.        mammalian) telomeric sequences are exposed.

Still another aspect of the present invention provides a geneticconstruct comprising an origin of replication and a first nucleic acidmolecule defining a human neocentromere or a functional derivativethereof or latent, synthetic or hybrid form thereof, a second nucleicacid molecule encoding a peptide, polypeptide or protein, wherein saidfirst and second nucleic acid molecules are flanked by a first set ofeukaryotic (e.g. mammalian, such as human) telomeric sequences which arein turn flanked by a second set of eukaryotic (e.g. yeast) telomericsequences wherein there are unique enzyme sites between the first andsecond telomeric sequences such that upon contact with a requiredenzyme, the second telomeric sequences are cleaved off to expose thefirst telomeric sequences.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic representation showing identification of a YACcontig spanning the marker centromere region. (A) Comparison of GTLbanding patterns of mardel 10 and normal chromosome 10. The pair of openarrows indicate the two breakpoints on a normal chromosome 10 ingenerating the marker chromosome (Voullaire et al., 1993). The long andshort arms of the marker chromosome are designated q′ and p′,respectively, to distinguish them from the q and p arms of the normalchromosome 10. Asterisk denotes the position of a cosmid 10pC38 that wasused to “tag” the q′-arm of stretched marker chromosomes in theANTI-CEN/FISH experiments. (B) A 4-megabase YAC contig (#082) from10q25.2 region that spans the marker centromere. The tilling path ofYACs #0 to #23 and their corresponding CEPH library addresses are shown.(C) FISH mapping of selected YAC clones from contig #082 using normalfluorescence microscopy and standard metaphase chromosomes prepared fromtransformed lymphoblast cells of patient BE. The distribution of FISHsignals (vertical axis) is shown as a percentage of the signals on onearm of the marker chromosome that is in excess of those found on theopposite arm of the chromosome. The total number of fluorescence signalsscored for each of the YAC clones is indicated in brackets.

FIG. 2 is a photographic representation showing ANTI-CEN/FISH analysisof the marker centromere. (A) Detection of α-satellite DNA using amixture of α-satellite DNA probes (red signals) under low stringencyconditions. Centromeres were counter-labelled with CREST#6 autoimmuneantibody (pale blue dots; or white when superimposed on a redbackground). Chromosomes were prepared from transformed lymphoblastcells of patient BE. The right-hand panel represents greenpseudo-coloring of DAPI images of chromosomes to provide a betterdefinition of chromosome outline. Only the signal for the antibody, butnot that for α-satellite, was seen on the marker centromere (arrowed).(B) Simultaneous labelling of stretched human metaphase chromosomes withCREST#6 (red) and anti-CENP-C antibody, Am-C1 (pale blue), with thewhite color indicating full coincidence of the two antibody signals. (C)Detection of CENP-C on the marker chromosome. Simultaneous labelling ofthe marker chromosome (arrowhead) with (a) Am-C1 (pale blue) and (b)CREST#6 (red). (c) Combined images of a and b, showing completecoincidence of Am-C1 and CREST#6 signals. (d) FISH analysis of the samecell as a–c using the 10pC38 cosmid probe (pale blue dots and greenarrows) to identify the marker chromosome. Some loss of ANTI-CEN signal,especially for the Am-C1 antibody was seen following FISH. (e) Greenpseudo-coloring of DAPI images. A colour photograph corresponding tothis figure is available upon request.

FIG. 3 is a photographic representation showing ANTI-CEN/FISH analysisof cosmid clones on stretched (A, a–f) and superstretched (B) metaphasechromosomes. (a–c) Examples of cosmid signals (white arrows) localizedto the q′-region of the marker centromere. (d–f) Examples of cosmidsignals (white arrows) localized to the p′-region of the markercentromere. Green arrows indicate positions of the 10pC38 cosmid DNA tagused to mark the q′-end of the marker chromosome. (B) Mapping of Y6C21onto a superstretched metaphase chromosome. Not included is the 10pC38q′-tag signal located further to the left of the chromosomal segmentshown. ANTI-CEN signals are in red, FISH signals are in pale blue, andoverlapping ANTI-CEN and FISH signals are in white. Each of the picturesis accompanied by DAPI images of chromosomes pseudo-coloured in green. Acolour photograph corresponding to this figure is available uponrequest.

FIG. 4 Localization of the anti-centromere antibody-binding domain. a,Relative positions of different cosmid and PAC clones within the YAC#082 contig, using YAC-3 as a reference. Cosmids are designated as YnCm,where ‘n’ denotes the YAC of origin and ‘m’ denotes the cosmid number.PACs 1–5 are five different PAC clones isolated from a human PAC library(Genome Systems Inc). “HC-contig” represents a group of overlappingcosmids that map tightly around the marker centromere in ANTI-CEN/FISHexperiments. A genomic map corresponding to the depicted YAC region wasderived from the DNA of patient BE and shown above the YAC map. S, SalI;K, KspI; N, NotI; Sf, SfiI. b, Cumulative scoring of FISH signals inANTI-CEN/FISH experiments for cosmids Y3C64, Y6C8, Y3C94, Y7C14, Y4C45,Y6C10, Y6C21, Y3C3, PAC5, Y13C1, Y13C8, and Y17C6. The distribution ofFISH signals (vertical axis) is those found on the opposite arm of thechromosome. The total number of fluorescence signals scored for each ofthe cosmid clones is indicated in brackets. c, Restriction mapping ofthe 80-kb region covered by the eight overlapping cosmids of theHC-contig. These eight cosmids were derived from four different YACs(YAC-3, YAC-4, YAC-6, and YAC-7) and provided independent confirmationof the map. Furthermore, the map agreed fully with the restriction mapof a 120 kb-insert PAC clone (PAC4) that spanned the entire HC-contigregion. E, EcoRI; R, EcoRV; N, NotI.

FIG. 5 is a representation showing restriction analysis of genomic DNAof patient BE and those of his normal parents using Y6C10 as probe. DNAwas resolved on a PFGE (A) or standard agarose gel (B and C). Samples 1,2 and 3 were fibroblast cultures of mother of BE, father of BE, andpatient BE, respectively. Sample 4 was a somatic hybrid cell line BE2C1-18-5F containing the marker chromosome. Fragment sizes are inkilobases.

FIG. 6 is a representation of the fill nucleotide sequence of theHC-contig DNA derived from normal human chromosome 10q 25.2 region.

FIG. 7 is a diagrammatic representation of the method used to retrofitYAC3 and YAC5.

FIGS. 8A to J are diagrammatic representations of the different vectorsused for cloning DNA as YACs by the conventional restriction/ligationmethods.

FIG. 9 is a diagrammatic representation of circular TAR summarising therecombination process.

FIG. 10 is a diagrammatic representation showing modification of TARvector.

FIG. 11 is a diagrammatic representation of the cloning of 10q25 humanneocentromere DNA from mardel (10) chromosome. This DNA is designatedNC-contig DNA to distinguish it from the HC-contig derived from thecorresponding region of the normal chromosome 10. (A) Structural map ofthe NC-contig region and flanking DNA. Arrows indicate the relativepositions and directions of primers used in PCR analyses (Table 3). Therestriction sites EcoRI, EcoRV, SrfI, and SftI and SftI are indicated byE, R, Sr and Sf, respectively. The position of the TAR “hook” CE-F2 isrepresented by the solid box. The hatched bar represents HC- orNC-contig. p′ and q′ refer to the short and long arms of mardel (10),respectively. (B) Circular TAR strategy using the vectorspVC39-Alu/C3-F2(+) and pVC39-Alu/C3-F2(−) for the direct cloning of theneocentromere DNA from mardel (10). The position of the Alu consensussequence hook is represented by the white box. Crosses denote the sitesof recombination between the TAR vector and the genomic DNA at the Aluand C3-F2 hooks during cloning. (C) Structural maps of the resultingcircular YACs 5f-52-E8 and 5f-38-F2 containing the neocentromere DNA ofthe mardel (10) chromosome. The DNA flanking the NC-contig isrepresented by stippled bars. (D) Structural maps of BAC/E8-1 andBAC/F2-14. Nt represents NotI and URA-BAC-neo represents theretrofitting vector BRV1 (Larionov et al., 1997).

FIG. 12 is a diagrammatic representation showing specific TAR ofHC-region from mardel 10.

-   -   The method was as follows: (1) Co-transformation into        YPH857; (2) Select HIS⁺ colonies; (3) screen for HC-region by        PCR; (4) Prepare high-MW DNA; (5) Digest with I-Sce1 to expose        hTELS; (6) Transfect HT 1080 cells; (7) Select for G418^(R);        and (8) analyse by PFGE and FISH.

FIG. 13 is a diagrammatic representation showing cloning in yeast asYAC/HAC.

FIG. 14 is a diagrammatic representation outlining TACT procedure.

FIG. 15 is a diagrammatic representation of TACT constructs.

FIGS. 16A(1)–16A(37), when joined at matchlines A—A through J′—J′,depict the full nucleotide sequence (SEQ ID NO:4) of the NC-contig DNAderived from mardel (10), which corresponds to the HC-contig DNA regionof the normal chromosome 10.

FIGS. 16B(1)–16B(34), when joined at matchlines A—A through G′—G′,depict partial nucleotide sequence of the BAC/F2-14 clone that isderived from a region immediately p′ of the NC-contig DNA (see FIG. 11D)(SEQ ID NOS: 5–29).

SUMMARY OF SEQ ID NOs. SEQ ID NO. DESCRIPTION 1 DNA primer 2 DNA primer3 Nucleotide sequence of HC-contig 4 Nucleotide sequence of NC-contig 5BAC-F2 contig 1 6 BAC-F2 contig 2 7 BAC-F2 contig 3 8 BAC-F2 contig 4 9BAC-F2 contig 5 10 BAC-F2 contig 6 11 BAC-F2 contig 7 12 BAC-F2 contig 813 BAC-F2 contig 9 14 BAC-F2 contig 15 15 BAC-F2 contig 33 16 BAC-F2contig 39 17 BAC-F2 contig 41 18 BAC-F2 contig 42 19 BAC-F2 contig 44 20BAC-F2 contig 47 21 BAC-F2 contig 47 fragment 1 22 BAC-F2 contig 47fragment 2 23 BAC-F2 contig 47 fragment 3 24 BAC-F2 contig 47 fragment 425 BAC-F2 contig 47 fragment 5 26 BAC-F2 contig 47 fragment 6 27 BAC-F2contig 47 fragment 7 28 BAC-F2 contig 47 fragment 8 29 BAC-F2 contig 47fragment 9 ABBREVIATIONS USED IN THE SUBJECT SPECIFICATION mardel (10):Marker chromosome from patient BE; comprises a rearrangement ofchromosome 10. HAC: Human artificial chromosome YAC: Yeast artificialchromosome MAC: Bacterial artificial chromosome PLAC: Plant artificialchromosome neocentromere: A centromere containing no substantialα-satellite DNA CENP: Centromere binding protein HC-contig: Region ofnormal chromosome 10 comprising neocentromere E8: q′ end/region ofmardel (10) neocentromere F2: p′ end/region of mardel (10) neocentromereBE: Patient from which mardel (10) identified TAR:Transformation-associated recombinant PCR: Polymerase chain reactionMarker neocentromere on mardel (10). neocentromere: NC-contig region ofmardel (10) chromosome comprising neocentromere

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention is predicated in part on the identification andisolation of nucleic acid molecules exhibiting neocentromericproperties. In accordance with the present invention, a neocentromere isconsidered a centromere which does not contain substantial α-satelliteDNA repeat sequences and, when activated, is capable of functioning as acentromere. The term “substantial” in this context means that thenucleic acid molecule does not contain detectable α-satellite by FISHanalysis under medium stringency conditions. The neocentromere maycontain a small number of highly diversed α-satellite DNA. In primates,α-satellite DNA is consider 171bph in length. An nucleic acid moleculecontaining an activated neocentromere or a neocentromere otherwisefunctioning as a centromere facilitates in accordance with the presentinvention, the nucleic acid molecule replicating, remainingextra-chromosomal and segregating with cell division. Reference hereinto “neocentromere” is taken to mean a centromere substantially devoid ofα-satellite DNA repeat sequences.

Accordingly, one aspect of the present invention provides an isolatednucleic acid molecule comprising a sequence of nucleotides which definesan eukaryotic neocentromere.

More particularly the present invention provides an isolated nucleicacid molecule comprising a sequence of nucleotides derived from aeukaryotic chromosome and encompassing a neocentromere which nucleicacid molecule when introduced into a compatible cell is capable ofreplicating, acting as an extra-chromosomal element and segregating withcell division.

The present invention is exemplified herein by the identification andcloning of a human neocentromere. This is done, however, with theunderstanding that the present invention extends to all eukaryoticneocentromeres such as from many plant, aviary, insect, fugal, yeast andreptilian chromosomes. The most preferred neocentromere, however, isfrom human chromosomes and their mammalian homologues.

The present invention is predicated in part on the identification of anunusual chromosomal marker in a patient designated “BE”. The chromosomalmarker is referred to as “mardel (10)” and results from a rearrangementof human chromosome 10. The mardel (10) marker is mitotically stableand, in accordance with the present invention, contains a functionalneocentromere at a location regarded as non-centromeric. Theneocentromere at mardel (10) is located between q24 and q26 onchromosome 10 and more particularly around q25. Even more particularly,the neocentromere maps to q25.2 on chromosome 10. The present inventionis exemplified by DNA cloned from the q24–q26 region of the mardel (10)chromosome as well as the corresponding region on normal humanchromosome 10. These DNA molecules contain a functional neocentromere.The present invention extends, however, to any neocentromere or anychromosome in mammalian and non-mammalian animals as well as plants,yeasts and fungi.

For convenience, the DNA clones from the mardel (10) chromosome as wellas from normal human chromosome 10 are summarized in FIG. 11. Theneocentromere located at or around 10q25 is located on a clonedesignated the “HC-contig”. DNA clones from mardel (10) are referred toas “E8” or the “NC-contig” which extends from the long arm (q′) ofmardel (10) towards the short arm (p′). Clone F2 extends further p′ fromE8 (see FIG. 11). It is emphasised, however, that the present inventionextends to any neocentromere on any human chromosome as well asneocentromeres on other mammalian and non-mammalian chromosomesincluding chromosomes from plants, insects, reptiles, yeast and fungi.

The present invention further contemplates a nucleic acid molecule orits chemical equivalent having a tertiary structure which defines ahuman neocentromere or a functional derivative thereof or a latent,synthetic or hybrid form thereof or its mammalian or non-mamalianhomologue.

Even more particularly, the present invention is directed to an isolatednucleic acid molecule having a sequence of nucleotides or their chemicalequivalents which directs a conformation defining a human neocentromereor a functional derivative thereof or a latent, synthetic or hybrid formthereof or its mammalian or non-mammalian homologue wherein thecentromere associates with centromere binding proteins (CENP)-A andCENP-C or antibodies thereto.

Reference herein to “latent” in relation to a centromere includesreference to a centromere not normally functional but neverthelessactivatable under certain conditions. A latent centromere may also beconsidered as a neocentromere provided it has no substantial α-satelliteDNA repeat sequences.

The size of the neocentromere in accordance with the present inventionmay range from about 50 bp to about 1500 kbp, from about 70 bp to about1000 kbp, from about 75 bp to about 800 kpb, from about 80 bp to about500 kbp, from about 85 bp to about 200 kbp, from about 90 bp to about100 kbp, from about 100 bp to about 1 kbp, about 120 bp to about 500 bp,about 180 bp to about 300 bp. In one particular embodiment, thecentromere is approximately 60–100 kbp. In another embodiment, thecentromere is about 80 kbp.

The nucleic acid molecule encompassing the HC-contig for humanchromosome 10 of the present invention set forth in FIG. 6 (SEQ ID NO:3). The nucleic acid molecule encompassing the NC-contig (part of E8)from mardel (10) is set forth in FIG. 16A (SEQ ID NO: 4). The nucleicacid molecule encompassing F2 of mardel (10) is set forth in FIG. 16B asseparate contigs (SEQ ID NOs: 5–29). The nucleic acid molecules have atertiary structure and the neocentromere is a conformation ofnucleotides within this tertiary structure. Accordingly, theneocentromere is not defined by a linear sequence of nucleotidesalthough this linear sequence directs the conformation which in turndefines the neocentromere. Although this aspect of the present inventionis exemplified using the nucleotide sequence set forth in FIGS. 6, 16Aand 16B, the subject invention extends to any sequence directing aconformation defining a centromere and hybridising to the sequence setforth in one or more of FIGS. 6, 16A and/or 16B under low stringencyconditions at 42° C. and/or which comprises a nucleotide sequence havingat least about 40% nucleotide similarity to one or more sequences setforth in FIGS. 6, 16A and/or 16B. Preferably, the percentage similarityis at least about 50%, more preferably at least about 60%, still morepreferably at least about 70%, even more preferably at least about80–90% or above such as 95%, 97%, 98% and 99%.

Another embodiment of the present invention is directed to YAC 3 and YAC5 encompassing the HC contig and flanking sequence as well as nucleotidesequences related to YAC 3 and/or YAC 5 at the homology, similarity orhybridization levels.

Reference herein to a low stringency at 42° C. includes and encompassesfrom at least about 1% v/v to at least about 15% v/v formamide and fromat least about 1M to at least about 2M salt for hybridisation, and atleast about 1M to at least about 2M salt for washing conditions.Alternative stringency conditions may be applied where necessary, suchas medium stringency, which includes and encompasses from at least about16% v/v to at least about 30% v/v formamide and from at least about 0.5Mto at least about 0.9M salt for hybridisation, and at least about 0.5Mto at least about 0.9M salt for washing conditions, or high stringency,which includes and encompasses from at least about 31% v/v to at leastabout 50% v/v formamide and from at least about 0.01M to at least about0.15M salt for hybridisation, and at least about 0.01M to at least about0.15M salt for washing conditions. These stringency conditions may bealtered dependent on the source of DNA and other factors.

The term “similarity” as used herein includes exact identity betweencompared sequences at the nucleotide level. Where there is non-identityat the nucleotide level, “similarity” includes differences betweensequences which nevertheless result in conformation defining afunctional neocentromere.

The nucleic acid molecule of the present invention may comprise anaturally occurring nucleotide sequence from a healthy human subject ormay comprise the nucleotide sequence from a human subject exhibiting oneor more chromosomal-dependent conditions such as a subject carryingmardel 10 chromosome or a chromosome conferring an equivalent or similarcondition or may carry one or more nucleotide substitutions, deletionsand/or additions relative to the naturally or non-naturally occurringsequence. Such modifications are referred to herein as “derivatives” andinclude mutants, fragments, parts, homologues and analogues of thenaturally occurring nucleotide sequence. Preferably, the derivatives ofthe present invention still define a functional neocentromere.

Reference herein to a “neocentromere” includes reference to a functionalneocentromere or a functional derivative thereof meaning that it iscapable of facilitating sister chromatid cohesion and chromosomalsegregation during mitotic cell divisions and/or is capable ofassociating with CENP-A and/or CENP-C and/or is capable of interactingwith anti-CENP-A antibodies or anti-CENP-C antibodies. Generally, andpreferably, the neocentromere is incapable of interacting with CENP-B oranti-CEP-B antibodies. Alternatively, the neocentromere may be a latentcentromere capable of activation by epigenetic mechanisms. Theneocentromere may also be a hybrid of other human, mammalian, plant oryeast neocentromeres. Synthetic neocentromeres provided by, for example,polymeric techniques to arrive at the correct confromation are alsocontemplated by the present invention. All such forms and definitions ofneocentromere are encompassed by use of this term.

Another aspect of the present invention provides an isolated nucleicacid molecule or chemical equivalent having the followingcharacteristics:

-   -   (i) comprises a nucleotide sequence or chemical equivalent        directing a conformation which defines a neocentromere or a        functional derivative thereof or a latent, synthetic or hybrid        form thereof or;    -   (ii) comprises a nucleotide sequence or chemical equivalent        substantially as set forth in one or more of FIGS. 6, 16A and/        or 16B or having at least about 40% similarity thereto or        capable of hybridising thereto under low stringency conditions        at 42° C.; and    -   (iii) comprises a neocentromere capable of associating with        CENP-A or CENP-C or antibodies thereto.

Preferably, the neocentromere is incapable of interacting with CENP-B orantibodies thereto.

In a particularly preferred embodiment, the centromere corresponds to ahuman genomic region which maps between q24 and q26 on chromosome 10,and in particular q25 on chromosome 10.

The nucleic acid molecule or its chemical equivalent of the presentinvention defining a conformational neocentromere or functionalderivative thereof or latent, synthetic or hybrid form thereof is usefulinter alia for the generation of artificial chromosomes such as humanartificial chromosomes (HACs), mammalian artificial chromosomes (MACs),yeast artificial chromosomes (YACs) and plant artificial chromosomes(PLACs). HACs are particularly useful since they are capable ofaccommodating large amounts of DNA and are capable of propagation inhuman cells. The HACs are non-viral in origin and, hence, are moresuitable for gene therapy by, for example, introducing therapeuticgenes. Furthermore, the HACs remain extra-chromosomal and, hence, haveno insertional/substitutional mutagenic potential. The essence of a HACis the presence of a neocentromere or latent, synthetic or hybrid formthereof which enables stable segregation during cell division. The HACalso remains extra-chromosomal and, hence, is more suitable for genetherapy. Reference to “extra-chromosomal” means that it does notintegrate into the main chromosome and, in effect, is episomal.

Accordingly, the present invention provides a genetic constructcomprising an origin of replication for a eukaryotic cell and a nucleicacid molecule encompassing a eukaryotic neocentromere or a functionalderivative thereof or a latent, synthetic, hybrid form thereof or itsmammalian or non-mammalian homologue flanked by telomeric nucleotidesequences functional in the cell in which the genetic construct is toreplicate and wherein said genetic construct when introduced into a cellis a replicating, extra-chromosomal element which segregates with celldivision.

More particularly, the present invention further contemplates a geneticconstruct in the form of an artificial chromosome comprising an originof replication for a mammalian, human, plant or yeast cell and a nucleicacid molecule encompassing a human neocentromere or a functionalderivative thereof or a latent, synthetic or hybrid form thereof or itsmammalian or non-mammalian homologue flanked by telomeric nucleotidesequences functional in the cell in which the artificial chromosome isto replicate.

Another embodiment provides a genetic construct in the form of anartificial chromosome comprising an origin of replication for amammalian, human, plant or yeast cell and a nucleic acid molecule havinga tertiary structure which defines a human neocentromere or a functionalderivative thereof or a latent, synthetic or hybrid form thereof or itsmammalian homologue flanked by telomeric sequences functional in thecell in which the artificial chromosome is to replicate.

Yet another embodiment is directed to a genetic construct in the form ofan artificial chromosome comprising an origin of replication for amammalian, human, plant or yeast cell and a nucleic acid molecule havinga sequence of nucleotides which directs a conformation defining a humanneocentromere wherein the centromere associates with CENP-A and/orCENP-C or antibodies thereto and does not contain substantialα-satellite DNA repeat sequences, said nucleic acid molecule flanked bytelomeric nucleotide sequences functional in the cell which theartificial chromosome is to replicate.

Still yet another aspect of the present invention relates to a geneticconstruct in the form of an artificial chromosome comprising an originof replication for a mammalian, human, plant or yeast cell and a nucleicacid molecule comprising a sequence of nucleotides which:

-   -   (i) directs a conformation which defines a neocentromere or a        functional form thereof or a latent, synthetic or hybrid form        thereof;    -   (ii) comprises a nucleotide sequence substantially as set forth        in one or more of FIGS. 6, 16A and/or 16B or having at least        about 40% similarity to the nucleotide sequences set forth in        FIGS. 6, 16A and/or 16B or is capable of hybridising to one or        more of these sequences under low stringency conditions at 42°        C.;        wherein the neocentromere is capable of associating with CENP-A        and/or CENP-C or antibodies thereto and wherein said nucleic        acid molecule is flanked by telomeric nucleotide sequences        functional in the cell in which the artificial chromosome        replicates.

In a preferred embodiment, the genetic construct is a HAC and compriseshuman telomeric sequences. In a particularly preferred embodiment, theHAC further comprises yeast artificial chromosome (YAC) arms and, hence,becomes a HAC/YAC shuttle vector capable of propagation in human andyeast cells. Preferably, the HAC/YAC contains a unique enzyme sitebetween yeast telomeric sequences and human telomeric sequences suchthat upon contact with the particular enzyme, the yeast telomericsequences are removed leaving the human telomeric sequences. Preferably,the unique enzyme site is a yeast specific enzyme site such as I-SceI.

According to this embodiment, there is provided a genetic constructdefining a HAC/YAC comprising an origin of replication and a nucleicacid molecule encompassing a human neocentromere or a functionalderivative thereof or a latent, synthetic or hybrid form thereof or amammalian or non-mammalian homologue thereof, said nucleic acid moleculeflanked by human telomeric sequences which are in turn flanked by yeasttelomeric sequences wherein a unique enzyme site is located between thehuman and yeast telomeric nucleotide sequences such that upon contactwith the enzyme, the yeast telomeric sequences are removed and the humantelomeric sequences are exposed.

More particularly, the present invention is directed to a geneticconstruct defining a HAC/YAC comprising an origin of replication and anucleic acid molecule encompassing a human centromere or a functionalderivative thereof or a latent, synthetic or hybrid form thereof or amammalian or non-mammalian homologue thereof wherein the neocentromereassociates with CENP-A and/or -C or antibodies thereto and does notcontain substantial α-satellite DNA sequences wherein said nucleic acidmolecule is flanked by human telomeric sequences which are in turnflanked by yeast telomeric sequences wherein a unique enzyme site islocated between the human and yeast telomeric nucleotide sequences suchthat upon contact with said enzyme, the yeast telomeric sequences areremoved and the human telomeric sequences are exposed.

Even more particularly, the present invention is directed to a geneticconstruct in the form of a HAC/YAC comprising an origin of replicationand a sequence of nucleotides which directs a conformation defining ahuman neocentromere or a functional derivative thereof or a latent,synthetic or hybrid form thereof or a mammalian or non-mammalianhomologue thereof wherein said neocentromere is capable of associatingwith CENP-A and/or CENP-C or antibodies thereto, said sequence ofnucleotides flanked by human telomeric sequences which are in turnflanked by yeast telomeric sequences wherein a unique enzyme site islocated between the human and yeast telomeric nucleotide sequences suchthat upon contact with said enzyme, the yeast telomeric sequences areremoved and the human telomeric sequences are exposed.

Preferably, the length of the nucleotide sequence is between about 30kpb and 1500 k/pb, and more preferably between 60 kbp and 1000 kpb.

In a particularly preferred embodiment, the unique enzyme site is ayeast specific enzyme site such as I-SceI.

The present invention extends to yeast cells and human cells carryingthe genetic constructs of the present invention and to proteins producedtherefrom.

The genetic constructs may also comprise marker genes and other uniquerestriction sites to facilitate insertion of adventitious DNA.Accordingly, the genetic constructs of the present invention may furthercomprise adventitious or heterologous DNA encoding a product ofinterest. Preferred products of interest include pharmaceutically usefulgenes such as genes encoding cytokines, receptors, growth regulators andthe like. Endogenous genes may also be replaced by wild-type genes ormodified genes.

The adventitious or heterologous DNA may also encode a molecule notsynthesised in a sufficient amount in a particular subject and hence theincreased copy number permits greater amounts of the molecule beingsynthesised.

Accordingly, the present invention contemplates a genetic constructcomprising an origin of replication and a first nucleic acid moleculedefining a human neocentromere or a functional derivative thereof orlatent, synthetic or hybrid form thereof or a mammalian or non-mammalianhomologous, a second nucleic acid molecule encoding a peptide,polypeptide or protein, wherein said first and second nucleic acidmolecules are flanked by a first set of human telomeric sequences whichare in turn flanked by a second set of yeast telomeric sequences whereinthere are unique enzyme sites between the human and yeast telomericsequences such that upon contact with said enzyme, the yeast telomericsequences are cleaved off to expose the human telomeric sequences.

Reference herein to segregate preferably means mitotically stablesegregation. Conveniently, stable segregation may be determined as thepresence of an artificial chromosome in 40–60% of daughter cells after4–6 months of continuous passage.

The present invention extends to other artificial chromosome analoguesto the HACs and HAC/YACs described above such as MACs and PLACs.

Another aspect of the present invention relates to peptides,polypeptides and proteins which bind, interact or otherwise associatewith the human neocentromere of the present invention or its mammalianand non-mammalian homologue. Preferably, the molecules are proteins,referred to as primary (1°) proteins. The 1° proteins bind to theneocentromere and secondary (2°) proteins bind to the 1° proteins beforeor after association with the neocentromere. The identification of thehuman neocentromere in accordance with the present invention provides amechanism for assaying 1° proteins and 2° proteins which may beimportant for screening chromosomes in, for example, genetic disorders.This is particularly the use in Down's Syndrome which results fromdefective chromosome segregation.

The 1° proteins are readily detected by, for example, a gel shift assay.The nucleic acid molecule of the present invention defining the humanneocentromere is digested, labelled and contacted with nuclear extractputatively containing the 1° proteins and resolved on a gel. When a 1°protein binds to a fragment carrying a binding portion of theneocentromere, the DNA fragment migrates in the gel at a slower rate dueto the bound protein.

The present invention extends to purified 1° proteins capable ofassociation with the subject centromere and to genetic sequencesencoding same and to antibodies thereto.

The neocentromeres of the present invention am readily identified andcharacterised using, for example, human fibrosarcoma cell lines. Forexample, DNA suspect of carrying a neocentromere, is introduced intofibrosarcoma cells in a linear form, generally together with a telomericsequence. The cells are then screened for the presence of replicating,extra-chromosomal and segregating elements, referred to as minichromosomes.

The present invention further encompasses eukaryotic cells carryingreplicating, extrachromosomal and segregation nucleic acid molecules.Preferably the eukaryotic cells are mammalian cells and most preferablyhuman cells. The nucleic acid molecules according to this aspect of thepresent invention are preferably as herein described. Particularlypreferred cells are HT-38, HT-47, HT-54, HT-190, HT-191, BAC/E8-1, andBAC/F2-14.

The present invention is further described by the following non-limitingFigures and Examples.

EXAMPLE 1 YAC and Cosmid Probes for FISH

YACs carrying specific STSs were identified (Moir et al., 1994) byPCR-based screening of YAC libraries prepared in pYAC4 vector at theCenter for Genetics in Medicine at Washington University (Brownstein etal., 1989) and at the CEPH (Albertsen et al., 1990). Cosmid DNA inserts(35–40 kb) were ligated to SuperCos I vector (Stratagene) and packagedwith Gigapack III Gold extract (Stratagene) according to themanufacturer's instructions. YAC probes were prepared by Alu-PCR oftotal yeast genomic DNA using primers 5′-GGATTACAGG(C/T)(A/G)TGAGCCA-3′[SEQ ID NO:1] and 5′-(A/G)CCA(C/T)TGCACTGCAGCCTG-3′ [SEQ ID NO:2]according to published method (Archidiacono et al., 1994). For probelabelling, 1 μg of the YAC PCR products or whole cosmid DNA isolated byCsCl centrifugation or Qiagen column was used. The DNA was labelled withBiotin-16-dUTP (Boehringer Mannheim) using a NICK translation kit(Boehinger Mannheim). A probe mix of 6–10 μg/ml of biotinylated probeDNA, 300 μg/ml of COT-1 DNA (Boehringer Mannheim), 500 μg/ml of carriersalmon sperm DNA and, where indicated, 10 μg/ml of biotinylated 10pC38tag DNA was ethanol precipitated, resuspended in a hybridization mix of50% v/v formamide in 2×SSC and 10% w/v dextran sulphate, denatured at95° C. for 5 min, preannealed for 30–60 min at 37° C. to suppressrepetitive sequences, before adding to slides. FISH of α-satellite andsatellite III probes was performed under low stringency as previouslydescribed (Voullaire et al., 1993).

EXAMPLE 2 Somatic Cell Hybrids and Other Cell Lines

Skin fibroblasts and transformed lymphoblast cell lines were establishedfrom patient BE (Voullaire et al., 1993) and from his normal parents.The presence of the mardel 10 chromosome in the patient cell lines wasconfirmed by FISH. In addition to these cell lines, two somatic cellhybrids were produced by fusing cultured fibroblast cells derived frompatient BE with the Chinese hamster ovary cell line CHO-K1 usingpolyethylene glycol. Hybrid cells were selected in a proline-free mediumfor the glutamic oxaloacetic transaminase-1 (GOT-1) gene located in10q24–q25 region. One of the hybrid cell lines, designated BE2C1-18-1f,was shown to contain the normal chromosome 10 but not the markerchromosome, while another hybrid cell line, designated BE2C1-18-5F,contained the marker chromosome but not the normal chromosome 10 ofpatient BE. The presence or absence of these chromosomes was establishedby karyotyping and ANTI-CEN/FISH probing. In addition, PCR analysis ofan STS (sequence tagged site) marker, AFM259xg5, which resided on YAC-3,confirmed the status of these chromosomes in the hybrids and excludedthe presence of submicroscopic fragments of the marker centromere regionwithin the genome of BE2C1-18-1f, or the presence of the correspondingregion of normal chromosome 10 within the genome of BE2C1-18-5f. Use ofthis STS marker also demonstrated that the mardel 10 chromosome hasoriginated from the patient's father.

EXAMPLE 3 Antisera

Antiserum CREST #6 was from a patient with calcinosis, Raynaud'sphenomenon, esophageal dysmotility, sclerodactyly and telangiectasia (aconstellation of symptoms commonly referred to as “CREST”; Moroi et al.,1981; Fritzler and Kinsella, 1980; Brenner et al., 1981). Western blotanalysis of this antiserum indicated that the primary antigens detectedwere human CENP-A and CENP-B. A specific anti-CENP-C polyclonalantibody, designated Am-C1, was produced by the inventors by expressinga partial mouse CENP-C polypeptide (amino acid #41 to 345) as aGST-fusion product in E. coli, followed by gel purification of theproduct and its use as an antigen for antibody production in rabbit.

EXAMPLE 4 Preparation of Standard Metaphase Chromosomes for FISHAnalysis

Actively replicating transformed lymphoblasts were incubated at 37° C.for 17 h in the presence of 0.1M final concentration of thymidine beforethey were centrifuged at 2000 rpm for 10 min, washed with pre-warmedRPMI, and incubated for a further 5–6 h. 15 min before harvesting,colcemid (10 μg/ml) was added. Cells were harvested according tostandard cytogenetic techniques using 0.075M KCl hypotonic solution for15 min at 37° C., followed by three fixative washes in ice coldmethanol/acetic acid 3:1, dropped onto clean glass slides, and storeddessicated at −20° C. until required

EXAMPLE 5 Preparation of Mechanically Stretched Chromosomes forANTI-CEN/FISH Mapping METHOD-I

This is an adaptation of the method described by Page et al. (1995).Colcemid (10 μg/ml) was added to actively dividing transformedlymphoblasts for 2–3 h, before the cells were centrifuged at 1500 rpmfor 10 min, washed in PBS, and resuspended in 0.075M KCl hypotonicsolution for 10 min at RT at a concentration of approximately 5×10⁴cells/ml; the use of fewer cells here gave better stretching of thechromosomes. 200–300 μl of this suspension were then cytocentrifugedonto clean microscope slides using a Cytospin 2 (Shandon) at 1000 rpmfor 5 min at high acceleration. The slides were immediately removed,placed flat in a shallow dish and very gently flooded with KCM(Potassium Chromosome Medium:120 mM KCl, 20 mM Nacl, 10 mM Tris-HCl, 0.5mM Na₂EDTA, 0.1% v/v Triton X-100) (Jeppesen et al., 1992). After 10 minat RT, immunofluorescence was performed without fixation (Earnshaw andMigeon, 1985; Earnshaw et al., 1989; Jeppesen et al., 1992; Jeppesen andTurner, 1993). KCM buffer was gently aspirated and 50 μl of CREST#6serum [diluted 1:50 in 1×TEEN (1 mM Triethanolamine HCl, 0.2 mM Na₂EDTA,25 mM NaCl), 0.1% v/v Triton X-100, 0.1% w/v BSA] was added to the cellarea of the slide and covered with a parafilm coverslip. The slides wereincubated for 30 mm at 37° C., then washed very gently by flooding in1×KB⁻⁽10 mM Tris-HCl (pH7.7), 0.15M NaCl, 0.1% w/v BSA), three rinses of3 min each at RT. The primary antibody was detected with TexasRed-conjugated Affini-pure Rabbit anti-Human IgG (H&L) (JacksonLaboratories) diluted 1:50 in 1×KB⁻. 50 μl was added to each slide,covered with a parafilm coverslip, and incubated for 30 min at 37° C.The slides were again gently washed by flooding in 1×KB⁻ for 2 min atRT, before they were fixed by flooding in 10% v/v formalin in KCM for 10min at RT, followed by three rinses of 3 min each in distilled water. IfFISH was not performed the slides were rinsed in PBS and mounted in DAP1(0.25 μg/ml) in DABCO antifade mountant. [In experiments where CREST#6and Am-C1 antisera were simultaneously used to label the centromere(FIGS. 2B and C), the above procedure was followed except for theaddition of Am-C1 diluted 1:100 together with CREST#6, and the Am-C1antibody was detected using 1:100 diluted Donkey anti-Rabbit DTAP(Jackson Laboratories)].

If FISH was to be performed on the slides, they were then given a secondfix in 3:1 methanol/acetic acid for 15 min at RT. The slides were airdried for at least 5 min and either processed for FISH or stored at −20°C. for up to several days before continuing. For FISH, the slides weredehydrated at RT in 70%, 90%, 100% v/v ethanol (2 min each) and airdried. Chromosomal DNA was denaturated in deionised 70% v/vformamide/2×SSC, pH 7.0 at 82° C. for 8 mm followed by immediatedehydration in 70%, 90% and 100% v/v ethanol at −20° C. for 2 min each,then air dried for at least 10 min. (This high temperature ofdenaturation was critical to obtain maximum FISH signals). An amount of15 μl of the prepared probe was added to each slide, covered with a 22mm² coverslip, and sealed with rubber cement. Slides were hybridizedovernight in a humid chamber at 37° C., then rinsed in 2×SSC at RT,followed by 3 washes of 0.1×SSC at 60° C. for 5 min each, rinsed againin 2×SSC, and immersed in a blocking agent of 5% non fat milk in 4×SSCfor 10 min at RT. Probe hybridization was detected by incubation withFITC-conjugated avidin at 37° C. for 30 min, followed by three washes of5 min each at RT in wash buffer (4×SSC, 0.05% v/v Tween-20). Signalswere amplified by incubating with goat anti-avidin D antibodies for 30min at 37° C., followed by three washes of 5 min each at RT in washbuffer, then with another layer of avidin-FITC for 30 min at 37° C.,before the slides were washed in wash buffer, rinsed in PBS, andcounter-stained with DAP1 (0.25 μg/ml) in DABCO mountant.

Method-II

The following method was modified from that of Haaf and Ward, (1994).Actively dividing lymphoblast cells were treated with 10 μg/ml colcemidfor 2–3 h, washed in PBS and resuspended in a hypotonic solutionconsisting of 10 mM Hepes (pH7.3), 30 mM glycerol, 1.0 mM CaCl₂ and 0.8mM MgCl₂, at a cell density of approx. 2.5×10²/ml. After 10 min ofhypotonic treatment at RT, 300 μl were cytocentrifuged (Shandon—Cytospin2) onto glass slides at 800 rpm for 4 min. The slides were immediatelyremoved from the centrifuge, dried for 15 sec, fixed in methanol at −20°C. for 20–30 min, rinsed in acetone at −20° C. for a few sec, thenwashed in 3 rinses of PBS at RT. Immunofluorescence staining was doneusing CREST#6 at a dilution of 1:50 in PBS. After incubation at 37° C.for 30 min, the slides were washed three times in PBS for 2 min each.This primary antibody was then detected by a further incubation for 30min at 37° C. with Texas Red-conjugated Rabbit anti-Human IgG diluted at1:50 in PBS. The slides were fixed in 10% v/v formalin in KCM for 10 minat RT, then washed in 3 rinses of distilled water and drained. BeforeFISH was performed, slides were fixed in methanol/acetic acid 3:1 for 15min at RT and air dried. Chromosomal DNA was denatured in 70% v/vdeionised formamide (pH7.0) in 2×SSC at 82° C. for 4–6 min. Afterdehydration in an ice cold ethanol series the slides were air dried, andused for FISH as described for Method I. Slides could be stored coveredin foil at RT after methanol/acetic acid fix for up to several weeksbefore FISH.

Both methods I and II were used to obtain the results shown in FIGS. 2B,2C, 3 and 4B.

EXAMPLE 6 Image Analysis

Hybridization signals for YAC mapping on standard metaphase preparationsutilized a normal fluorescence microscope. Images for the ANTI-CEN/FISHexperiments were analyzed on a Zeiss Axiolab fluorescence microscopeequipped with a 100× objective and a cooled CCD camera (PhotometricsImage Point) controlled by a Power Mac computer. Gray scale images werecaptured separately using a LUDL filter wheel and controller for TexasRed, FITC and DAPI. These images were pseudocoloured and merged usingIPlab Spectrum software from Signal Analytics Corporation. A number ofdifficulties were commonly associated with the ANTI-CEN/FISH technique:(a) the deliberate “stretching” of the chromosomes, whilst increasingthe resolution of mapping, sometimes caused serious distortion to thechromosomes, often making them quite dysmorphic; (b) FISH treatmentfollowing the ANTI-CEN-labelling often significantly reduced theANTI-CEN signals; (c) more highly stretched chromosomes (which wouldpotentially give better mapping resolution) generally gave weakerANTI-CEN signals; and (d) the ANTI-CEN signal on the mardel 10centromere was usually weaker than those of the other human chromosomes.Thus, a cell would only be considered informative and used for scoringif both the p′- and q′-arms of the mardel 10 chromosome were discernibleand separated by a discrete ANTI-CEN signal. In addition, FISH signalsfor both the test probe and the 10pC38 cosmid tag (used to identify theq′-arm of, and thus orientate, the marker chromosome) must be clearlypresent. Using these criteria, the overall frequency of informativecells was found to be approximately 1 in every 20–30 metaphasesanalyzed.

EXAMPLE 7 Restriction Analysis of Patient DNA

High-molecular weight genomic DNA was extracted from cultured fibroblastcell lines of patient BE and those of his parents and digested withdifferent enzymes to generate restriction fragments ranging from <1 kbup to ˜1 Mb. The digested DNA was resolved either on a standard agarosegel or by pulsed-field gel electrophoresis (PEGE) using a Bio-RadCHEF-XA Mapper. For filter hybridization, 50–100 ng of whole cosmid orPAC DNA was labelled by random priming. The labelled probe was thenadded to 2 ml of hybridization buffer (0.5M Na₂HPO₄, 7% w/v SDS, 1% w/vBSA, 1 mM EDTA, pH. 7.0) containing 500 μg of human placental DNA(Sigma). The mixture was boiled for 5 min, then placed in a 65° C. waterbath for preannealing of repetitive DNA for 90 min. The preannealedprobe mix was then added to prehybridizing filters and hybridizedovernight at 65° C. Post-hybridization washes were at a final stringencyof 0.1×SSC, 0.1% w/v SDS at 68° C.

EXAMPLE 8 Identification of a YAC Region Spanning the Marker Centromere

The initial search for DNA sequences spanning the centromere of themardel 10 chromosome was based on fluorescence in situ hybridization(FISH) of existing cosmid and YAC clones (Moir et al., 1994; Zheng etal., 1994) that have been mapped to the q24–q26 region of the normalhuman chromosome 10 where the new marker centromere was formed(Voullaire et al., 1993) (FIG. 1A). This search led to theidentification of a 4 megabase YAC contig (designated #082) that spannedthe marker centromere region (FIG. 1B). FIG. 1C graphically presents theFISH mapping results with selected YACs from this contig. As can beseen, two of the YACs (YACS-1 and YAC-2) mapped to the q′-side of themarker centromere, whereas the remaining YACs mapped to the p′-side ofthe centromere. The low signal level observed for YAC-3 was due to alarge proportion of this probe hybridising directly on the centromereitself. These results, therefore, provided evidence that YAC contig #082spanned the marker centromere, and that the centromere region was likelyto be within YAC-3, where the “cross-over” between the q′ and p′ signalsoccurred.

EXAMPLE 9 Development of Improved ANTI-CEN/FISH Methods for theSimultaneous Detection of Marker Centromere and Single-Copy Cosmid DNAProbes

Although normal fluorescence microscopy and FISH analysis of standardmetaphase chromosomes were adequate for the initial identification ofthe YAC contig spanning the marker centromere, methods withsignificantly higher sensitivity and resolution were needed to allowfurther walking into the marker centromere DNA. Three requirements haveto be satisfied by these methods: (a) the metaphase chromosomes have tobe extended to offer much greater mapping resolution, (b) thecentromeres have to be more precisely defined than that offered by acytogenetic constriction, and (c) the methods should allow simultaneousvisualization of both the centromere antibody and FISH signal. Twopublished methods were explored (designated here as ANTI-CEN/FISHmethods) based on extending metaphase chromosomes by mechanicalstretching and labelling of the neocentromere by autoimmune antibodies(Haaf and Ward, 1994; Page et al., 1995). Since these methods wereoriginally established for the labelling of normal centromeres and forFISH analysis of highly repeated DNA, they were modified (see Example 4)to allow detection of the generally reduced ANTI-CEN signal of thesubject marker neocentromere and the lower FISH signals resulting fromthe use of single-copy cosmid DNA probes.

With the improved detection methods, the status of α-satellite andsatellite III DNA on the marker neocentromere was reassessed, since thiswas previously determined using standard microscopy and FISH (Voullaireet al., 1993). FIG. 2A shows the result of antibody labelling usingCREST#6 and FISH using α-satellite DNA, and indicated the absence ofdetectable signal on the marker centromere. The same result was obtainedwhen the experiments were repeated without ANTI-CEN-labelling, rulingout the possibility that the anti-centromere antibody might haveobscured any weak FISH signals. Similar results were obtained withsatellite III DNA. Since in separate reconstruction experiments, it waspossible to demonstrate the sensitivity of the procedure in detecting asingle-copy DNA probe of less than 1.5 kb, and making the reasonableassumption that the low-stringency hybridization conditions used for theα-satellite and satellite III DNA which, by virtue of the useof >100-fold excess of probes and the strong hybridisation of theseprobes to all the other centromeres, would have allowed the detection ofany related sequences, it can be concluded that these satellite areabsent,

EXAMPLE 10 Co-Localization of CENP-C and CENP-A on the MarkerNeocentromere

To test if CENP-C is present on the marker centromere, a specific rabbitpolyclonal antibody was prepared against a recombinant product of mouseCENP-C. This antibody, designated Am-C1, reacted strongly with thecentromere of rodent and human chromosomes. FIG. 2B shows results forthe labelling of stretched human metaphase chromosomes using thisantibody simultaneously with the CREST#6 autoimmune antibody. As can beseen, irrespective of the degree of chromosome stretching, the signalsfor the two antibodies coincided fully on all the centromeres. Thelocalization of these two antibodies on the marker chromosome wasfurther determined by employing the 10pC38 cosmid tag in anANTI-CEN/FISH experiment to identify the marker chromosome. The resultsindicated that both the antibody signals were clearly present and againcoincided completely on the marker centromere (FIG. 2C, a-e). AlthoughCREST #6 was known to bind CENP-A and CENP-B, indirect evidence suggeststhat binding to the marker centromere presumably occurred via CENP-Asince the presence of the marker centromere was previously demonstratednot to bind CENP-B (Voullaire et al., 1993). The above results,therefore, established the localization of CENP-C, and probably CENP-A,on the marker centromere.

EXAMPLE 11 Localization of the Anti-centromere Antibody-Binding Domain

For further walking into the marker centromere region, cosmid librarieswere prepared from total yeast genomic DNA containing YACs-2, -3, -4,-6, -7, -13, and -17. Cosmid clones containing human DNA inserts wereisolated by hybridization with human COT-1 DNA using low stringency. Allresulting cosmids were screened by standard FISH to confirm theirlocalization to the expected marker centromere and normal chromosome 10regions, and to eliminate clones that might have originated from othergenomic sites due to chimeric YACs. Positive clones were then analyzedfurther with the ANTI-CEN/FISH methods, using CREST#6 to label thecentromere. FIG. 3 a (I and II) show examples of cosmid signals thatmapped to the q′- and p′-side, respectively, of the marker centromere inthe ANTI-CEN/FISH experiments. The cosmid tag (clone 10pC38) was used inthese experiments to define the q′ arm of the marker chromosome. Forcosmid walking, we concentrated on clones derived from YAC-3 since FISHmapping of YAC contig #082 indicated that the marker centromere regionwas likely to be within this YAC. FIG. 4 a shows a restriction map ofthe region covered by this and surrounding YACs and compares this mapwith a genomic map derived from patient BE. The relative positions of aseries of cosmid clones (including five independent PACs) were alsodetermined and placed on the YAC map. FIG. 4 b presents theANTI-CEN/FISH results obtained with a number of the cosmid clones andone of the PAC clones. Clones Y3C64, Y6C8, and Y3C94 localizedpreferentially to the q′-side, while Y13C1+C8 and Y17C6 localizedpreferentially to the p′-side of the marker centromere, suggesting thatthe nucleus of the antibody-binding domain is situated between these twocosmid clusters. Within this central region, a group of cosmid clonescomprising the HC-contig (FIG. 4 a) was found to map closely around theANTI-CEN signal. FIG. 4 c shows a restriction map for eight differentoverlapping clones from this HC-contig. The chromosomal positions offive of these overlapping clones were analyzed in detail usingANTI-CEN/FISH. FIG. 4 b shows the cumulative results for more than 60informative chromosomes for each of these five probes. The resultsindicated that Y7C14 mapped preferentially q′- of the antibody-bindingdomain, while the remaining four clones (Y4C45, Y6C10, Y6C21 and Y3C3)mapped preferentially to the p′-side. In addition, the results for PAC5(a 75 kb-insert PAC clone that overlapped with the p′-end of PAC4 byapproximately 5 kb; see FIG. 4 a) provided further evidence for theemergence of the HC-contig region onto the p′-arm. Based on theseresults, we conclude that the eight contiguous cosmid clones within theHC-contig shown in FIG. 4 c, which together constitute ˜80 kbp of DNA,have defined the nucleus of the antibody-binding domain of the markercentromere.

From the above ANTI-CEN/FISH results, it was difficult to determine ifthe sequences of the HC-contig and its surrounding DNA, both originallyderived from a normal individual, were part of the marker centromereDNA, or whether these sequences simply flanked a transposed centromereDNA with an unrelated nucleotide composition. However, supportingevidence from the ANTI-CEN/FISH experiments suggested that the DNA ofthe HC-contig region appeared to be a part of the marker centromere.This came from the mapping of Y6C10 and Y6C21 onto superstretchedchromosomes that were occasionally detected in the slide preparations.An example of such mapping is shown in FIG. 3 b using Y6C21. As can beseen, whilst a significant portion of Y6C21 hybridized to the p′-side ofthe CREST signal on the highly extended chromosome, a substantialportion of the cosmid DNA also overlapped directly with the CRESTsignal. This suggests that at least part of the HC-contig regionactually comprises the same DNA sequence as the marker centromere. Thispossibility was further investigated by detailed genomic mapping.

EXAMPLE 12 The Marker Centromere DNA has a Similar or Identical SequenceOrganization as the HC-Contig

The genomic organization of the HC-contig region was compared with thatof the corresponding DNA region of the mardel (10) chromosome. Threeoverlapping cosmids (Y7C14, Y6C10, and Y4C7, the latter beingessentially the same as Y6C21; FIG. 4C) from the HC-contig were used asprobes to analyze the restriction patterns of genomic DNA prepared frompatient BE and those of his karyotypically normal parents. FIG. 5 showsexamples of the band patterns obtained with Y6C10, while Table 1summarizes the results for all the enzymes tested with Y7C14, Y6C10 andY4C7. The detection of a single band on PFGE gels with a number of theenzymes indicated that the cosmid DNA sequences were unique within thehuman genome (SfiI, SalI, KspI, KpnI and BclI in FIG. 5A; Table 1). Thedetection of a single on PFGE gels with a number of the enzymes (ClaI inFIG. 5A; Table 1) could be explained by differential methylation ofdifferent restriction sites found in this region (Nelson and McClelland,1991); the reproducibility of these multiple band patterns ruled outincomplete digestion as a possible cause. The multiple bands detectedwith the more frequent cutting enzymes on a standard gel (FIG. 5B andTable 1) were a result of the presence of cleavage sites present withinthe probe DNA, since similarly digested cosmid DNA electrophoresed nextto the genomic DNA yielded identical patterns for all the bands notcontaining cosmid vector sequences. In all, 37 enzymes were used togenerate more than 160 different fragments for the three cosmid probes(Table 1). The results indicated that, except for a polymorphic fragmentfound in one of the parents, an identical banding pattern was present inthe genomic DNA of patient BE and those of his parents. Furthermore,when the restriction patterns obtained for the genomic DNA of patient BEwere compared with those of the somatic hybrid cell line BE2C1-18-5F,which contained the marker chromosome but not the normal chromosome 10,no detectable difference was seen between the two DNA preparationswithin the HC-contig region (FIG. 5C).

In addition to Y7C14, Y6C10 and Y4C7, a host of other probes from withinor surrounding the HC-contig have been tested, each with an average of12 different informative enzymes. These probes included PAC4 (whichspanned the entire HC-contig region shown in FIG. 4C), Y3C64, Y3C109,Y6C6, Y6C8, Y3C94, PAC1, Y3C90, Y4C4, Y4C8, Y4C13, Y4C33. The resultsagain indicated identical restriction enzyme patterns between patient BEand normal DNA. Thus, through the analysis of a relatively large numberof probes covering about 500 kb of YAC-3 around the HC-contig region,and the use of a high density of restriction enzymes that generated arange of fragments from <1 kb to ˜1 Mb, it was evident that the markercentromere DNA and a substantial stretch of its adjoining regions showedno detectable difference against the corresponding genomic region of thenormal chromosome 10.

Since a potential limitation of the above Southern blot analyses wasthat highly repeated sequences were not detected because of thepreannealing step used in the hybridisation procedure, a differentapproach was employed to compare the DNA of the marker chromosome andthat of the normal chromosome 10. In this approach, oligonucleotideprimers from different regions of the HC-contig were used to prepare aseries of PCR fragments from the BE2C1-18-5F and BE2C1-18-1F hybrid celllines. Electrophoretic comparison of such fragments, which randomlycovered approximately 40 kb of the HC-contig, indicated no detectabledifference between the two chromosomes and provided independent supportfor the results obtained in the Southern blot analyses. Thus, it can beconcluded that the sequence organization of the marker centromere regionis similar, if not identical, to that found in the HC-contig region ofthe normal chromosome 10.

EXAMPLE 13 Implications for Centromere Study and Mammalian ArtificialChromosome Construction

The mammalian centromere has been difficult to study due to the massiveamount of repetitive DNA normally associated with it. By avoiding suchrepetitive DNA and analyzing the unusual centromere found in the presentmarker chromosome, the inventors have created a much more tractablesystem for centromere studies. The present analysis has already shedsome light on the important question of DNA sequence versusconformational requirement of a centromere, and on the intriguingconcepts of latent centromeres and epigenetic mechanisms. One urgentapplication of this DNA is to use it to identify the primary protein(s)which binds to the centromeric DNA. Another important application of themarker centromere DNA is in the construction of mammalian artificialchromosomes. Such artificial chromosomes offer a potentially powerfulvehicle for the structural and functional analysis of chromosomes, forthe genetic manipulation of plants and animals, and for the stabletransmission of therapeutic genes in human gene therapy. The artificialchromosomes require a functional mammalian centromere, and the markercentromere DNA element of the present invention now provides a suitablecentromere especially because of its relatively small size in theabsence of α-satellite DNA and its cloning stability, as indicated bythe cosmid, YAK and BAC clones of the HC-contig and NC-contig.

EXAMPLE 14 Sequence Analysis

FIGS. 6, 16A and 16B show partial nucleotide sequences for the HC-contig(SEQ ID NO: 3) NC-contig [SEQ ID NO: 4] and F2 (BAC/F2–14) [SEQ ID NO:5–29] regions, respectively.

EXAMPLE 15 Human Artificial Chromosome (HAC)

The following are examples of the different approaches being used in theinventors' laboratory for the production of a HAC:

Retrofitting of HC-Contig DNA from Normal Chromosome 10

This procedure aims to produce HACs of 100 kb to >1 Mb using the regionof the normal chromosome 10 containing and surrounding the HC-contigDNA. The generation of a HAC by this approach will provide crucial proofthat this normal DNA region can be reactivated to form a functionalcentromere.

A retrofitting procedure suitable for introducing human telomeres toboth ends of any YAC prepared in the pYAC4 vector in the yeast hoststrain AB1380 has been previously described (Larin et al., 1994; Tayloret al., 1994, 1996). YACs (in particular YAC-3 and YAC-5) spanning thenormal HC-contig region are used for retrofitting by plasmid constructsdesigned to recombine with their pYAC4 vector arms (FIG. 7). Theconstruct pLGTEL 1 is used to target the left arms of the YACs. Thisserves to add a LYS2 yeast selectable marker, gpt element for ultimateselection in mammalian and avian cell culture, and a human telomere. Theright arm of the YACs are targeted by homologous recombination withpRANT 11 to produce a final construct where additional markers areintroduced along with a second human telomere to cap the construct.Specifically, an ADE2 yeast marker is added and the URA3 gene of the YACis disrupted, serving a useful role in negative selection of theconstruct. A neomycin (neo) resistance gene shown to function inmammalian and avian cells is also introduced. The finished constructsare transfected into different cultured cell lines, including HT1080 (ofhuman sarcoma origin) (Larin et al., 1994; Rasheed et al., 1974), DT40(a recombination-proficient chicken cell line) (Dieken et al., 1996),and BE2CI-18-5f (a human/hamster somatic hybrid cell line containing themardel (10) chromosome but not the normal chromosome 10).

In Vitro Cloning of HC-Region into YAC/HAC Vectors

The different vectors used for the cloning of the normal and mardel (10)centromeric DNA in the preparation of HACs are summarised in Table 2.

A number of different YAC cloning strategies are employed:

Conventional YAC cloning approach FIGS. 8A–D show the different vectorsused for cloning DNA as YACs by the conventional restriction/ligationmethods. These YACs can then be shuttled into mammalian cells and testedfor HAC function.

ALU-ALU circular TAR cloning approach. Transformation-associatedrecombination (TAR) in the yeast S. cerevisiae, is a method forconstructing linear and circular YACs from mammalian DNA (Larionov etal., 1996a, 1996b). The recombination process is shown in FIG. 9.Briefly, the technique involves the use of a vector (pVC39-AAH2, FIG.8E) lacking an autonomous replicating sequence (ARS) but containing afunctional yeast centromere (e.g. CEN6) and selectable marker (e.g.HIS3), and two ALU DNA hooks to trap mammalian DNA by recombination atALU sequences after co-transformation of linearized vector and highmolecular weight DNA into yeast spheroplasts and followed by selectionon medium lacking histidine. The key to the process is that themammalian DNA provides an ARS (11-bp sequence found frequently inmammalian DNA) which allows the HIS⁺/CEN vector to replicate as acircular YAC. These YACs are very stable and range in size from 100 kbto greater than 600 kb (Larionov et al., 1996b).

pVC39-AAH2 vector is used to clone DNA from hybrid BE2CI-18-5f to makeYACs with an average insert of 250 kb. This TAR vector is furthermodified to create pAAH-TCNa (FIG. 8G) so that it has the ability toshuttle between yeast and mammalian cells (as outlined in FIG. 10),including the potential to expose human telomeres (TEL) at each end of acloned fragment using a unique restriction site I-SceI.

Semi-specific and specific circular TAR. A modified circular TAR methodutilising two specific 5′C and 3′C DNA hooks (300–700 bp in size) may beused to clone a specific human DNA at a frequency of 3/1000 HIS⁺transformants. The inventors prepared the vectors pVC39-ALU/C3-F2(+/−)and pTCN-TCS (Table 2) to perform serni-specific and specific TARcloning, respectively.

The Semi-specific TAR methodology is a modification of a specificcircular TAR strategy which permits the site directed isolation oftarget chromosomal DNA. Furthermore, in accordance with the presentinvention, the methodology described herein enables the site-specificcloning of target chromosomal DNA from total genomic DNA as a circularYAC at relatively high frequencies and without the need for theconstruction and extensive screening of complex libraries made fromgenomic DNA.

In a preferred embodiment of the present invention, the methodologyemploys a single specific DNA hook which flanks the mardel (10)chromosome and a less specific Alu-hook to trap the other side of thetarget DNA.

In initial experiments, a unique repeat DNA-free, 1.4 kb EcoRI fragment(designated C3-F2) was identified from the p′ side of the 80-kbHC-contig (FIG. 11A) (du Sart et al., 1997). This fragment was subclonedinto the centromere-based yeast circular TAR vector, pVC39-AAH2, byreplacing the existing BLUR13 Alu (Larionov et al., 1996b) to create thepVC39-ALU/C3-F2 constructs. As the specific orientation of the C3-F2sequence on the chromosome was not known, the fragment was cloned in twodifferent orientations, for which the (+) orientation (FIG. 11B) wasexpected to trap the genomic region to the left of C3-F2, while the (−)orientation was expected to trap the region to the right. Bothconstructs were used in yeast transformation.

As a source of genomic DNA containing the neocentromere, a somatichybrid cell line, BE2C1-18-5f (du Sart et al., 1997), containing themardel 10 chromosome but not the normal human chromosome 10 was used. 5μg of high-molecular-weight DNA from this cell line and 1 μg ofpVC39-ALU/C3-F2(+) or pVC39Alu/C3-F2(−) (linearized with SmaI to exposethe 0.21-kb Alu and 1.4-kb C3-F2 hooks) were co-transformed into 10⁹(previously prepared and stored frozen) spheroplasts of S. cerevisiaeYPH857 which carries a HIS3 gene deletion, (Sikorski and Hieter, 1989)and grown on SD, without HIS medium, (Larionov et al., 1996a;b) to yieldbetween 10 and 100 HIS⁺ colonies. Control experiments in which YPH857was transformed with vector alone did not produce any colonies,indicating that the C3-F2 fragment lacked ARS-like sequences. Twenty TARexperiments were performed and HIS⁺ colonies were picked into 96-welltrays containing YPD medium (supplemented with 50 μg/ml ampicillin and15 μg/ml tetracycline), grown at 30° C. with aeration for 24 h andstored in 20% (v/v) glycerol at −70° C. Total yeast DNA was prepared inpools of 48 (Kwiatkowski jr et al., 1990) and screened by PCR with theprimers norm 5 and norm 7 (Table 3) which are located 30-kb q′ of C3-F2(FIG. 11A). Two desired positive clones, designated 5f-52-E8 and5f-38-F2, which contained the neo-centromere DNA derived from mardel 10and mardel (10) and the DNA immediately p′ of the neocentromeric DNA,respectively, were identified. For subsequent studies, these clones weregrown on SD without HIS medium and single colonies were re-isolated forcharacterization.

Initially, the sequence nature and sizes of the 5f-52-E8 and 5f-38-F2insert DNA were determined. High-molecular-weight DNA was prepared inagarose blocks and digested with an enzyme (SrfI) that linearized withYAC (FIG. 11A). The linearized DNA, as well as uncut intact DNA, wereresolved by pulsed-field gel electrophoresis (PFGE), transferred onto anylon membrane and probed with radiolabelled PAC4, a P1-derivedartificial chromosome clone containing a 120-kb insert that spans theentire HC-contig from normal chromosome 10, (du Sart et al., 1997)following preannealing with human placental DNA to suppress repetitiveDNA. The intact 5f-52-E8 and 5f-38-F2 remained trapped in theelectrophoretic wells and the linearized DNA migrated into the gel anddemonstrated a size of approximately 110 kbp and 80 kbp, suggestinginsert sizes of about 105 kbp and 75 kbp, respectively (given that thevector size is 5.9 kb).

Despite the use of a genomic DNA source previously shown bysequence-tag-site (STS) analysis to be free from normal chromosome 10material, it is desirable to independently confirm the mardel(10)-origin of the 5f-52-E8 YAC clone. This was achieved using a set ofprimers (norm 17 and 18; FIG. 11A) that detected avariable-number-tandem repeat (VNTR) region within theHC-contig/neocentromere region. The results clearly indicated thepresence of a 1.4-kb PCR product that was specific for the mardel (10)chromosome (Table 3).

PCR was used to further compare the 5f-52-E8 DNA with the previouslycloned HC-contig sequence derived from normal chromosome 10. PCRproducts with sizes ranging between 0.2 and 15.9 kb were generated bystandard PCR or with the Expand Long Template PCR system(Boehringer-Manneheim). Products greater than 1 kb were digested withfrequent cutting enzymes, RsaI and BsiXI, and their fingerprints werecompared by agarose gel electrophoresis. The results, shown in Table 3,indicated the absence of any detectable difference between the 5f-52-E8DNA and those of the corresponding regions of the normal chromosome 10(in somatic cell hybrid BF2C1-18-1f) and the neocentromere region ofmardel (10) (in somatic cell hybrid BE2C1-18-5f. These results alsodemonstrated that the YAC 5f-52-E8 spanned at least 75 kb of theHC-contig region (FIG. 11C), consistent with the size determined byPFGE. Furthermore, the ability of all the internal primers to amplifyDNA from 5f-52-E8 strongly suggested that the YAC was not chimeric. Thisresult was confined by isolating DNA from four single-colony isolates of5f-52-E8, digesting these with EcoRI and EcoRV, and probing withradiolabelled PAC4. The hybridization patterns obtained with theseenzymes were consistent with those established in the previous study (duSart et al., 1997). Thus, this analysis, based on cloned DNA deriveddirectly from mardel 10, has provided confirmation that theneocentromere DNA region is structurally identical to that of thecorresponding HC-contig region of the normal chromosome 10 (du Sart etal., 1997).

The circular YACs 5f-52-E8 and 5f-38-F2 were further retrofitted withthe yeast-bacterial-mammalian cells shuttle vector BRV1 as previouslydescribed (Larionov et al., 1997). The resulting BAC clones weredesignated BAC/E8-1 and BAC/F2-14, respectively (FIG. 11D).

The specific TAR strategy is outlined in FIG. 12 and uses uniquefragments from the HC-contig region, such as the ends of PAC4 (a 120kb-insert PAC clone containing the HC-region) to create the YAC/HACshuttle vector pTCN-TCS. An example of a YAC/HAC construct containingthe HC-contig region of normal chromosome 10 is shown in FIG. 13.

Completed constructs are transfected into different cultured mammalianor chicken cells (see above) by lipofection using Transfectam or DOSPER.

In Vivo “Cloning” of HC-Region into HAC Vectors

This strategy employs a technique known as Telomere AssociatedChromosomal Truncation (TACT) (FIG. 14). The technique is based on theprinciple that cloned mammalian telomeric DNA when reintroduced into amammalian cell can seed the formation of a new telomere at anintrachromosomal location. If the introduced telomeric DNA is targetedto a known site through homologous recombination, integration at thatlocation and subsequent truncation of distal sequences on the originalchromomosomal arm can result (Brown et al., 1994; Farr er al., 1995).This technique is employed in our own study to truncate the mardel 10chromosome on either side of the HC-contig/core centromeric DNA elementto produce in vivo a stable HAC of minimal size.

FIG. 15A shows an example of TACT-construct used in our study. Keyfeatures of this construct are: (a) Cloning of the pericentric humangenomic DNA in both orientations (+/−). This is necessary since we donot know the chromosomal orientation of this DNA. This DNA is used totarget the human telomeric sequences to locations on either side of theHC-contig region on mardel 10. Genomic DNA is derived from severaldifferent sources including Y2C24, Y3C64, Y3C109, Y3C94, Y13C12, Y13C15,Y17C6, Y17C8. The resulting truncation derivatives produced using thesegenomic DNAs will vary in size accordingly. (b) The termini contain 2.4kilobases of tandem repeat human telomeric DNA (htel). This DNA has beenshown previously to act as a substrate for mammalian telomerase to allowseeding of a complete telomere tens of kilobases in length. (c) Thehygromycin (Hyg) resistance gene allows for positive selection ofmammalian cell lines containing construct sequences integrated into thegenome. This is the initial screening procedure. In addition, someconstructs contain the neomycin phosophotransferase gene (Neo) ratherthan Hyg. (c) The Herpes simplex thymidine kinase (TK) gene is used fornegative selection against non homologous integration events into thegenome. Those cell lines containing the TK gene can be selected againstby adding the nucleoside analogue gancyclovir.

FIG. 15B shows another example of TACT-construct used in our study. Inaddition to the features of the linearised construct shown in FIG. 15A,specific additional features are: (a) The incorporation of tandemtelomeric blocks (htel.htel) since others have shown these to have thehighest seeding efficiency of new telomeres in mammalian cells. (b) Theincorporation of yeast selectable marker (eg. URA3), DNA origin ofreplication (eg. ARS), and centromere (eg. CEN6), to allow transfer andmaintenance of the resulting truncation derivatives into yeast. Thisshould facilitate further characterisation and manipulation, such as theintroduction of therapeutic genes for gene therapy purposes. (c) Therelocation of the TK gene adjacent to the genomic DNA to increase theeffectiveness of the negative selection system. (d) The human growthhormone (GH) gene has been included to allow proof of principle thathuman genes can be introduced into a HAC and expressed under the controlof endogenous regulatory elements. This is essential for gene therapyapplications of the resulting HAC. (e) A CMV promoter upstream of a P1phage loxP site (CMV/loxP) has been included to allow introduction oflarge human genes into a HAC in vivo. A plasmid containing a gene ofinterest, a second loxP site and a promoterless selectable marker geneis introduced into a mammalian cell line containing the HAC. Transientexpression of CRE recombinase results in recombination between the twoloxP sites within the cell, thereby integrating the introduced plasmidinto the HAC and placing the selectable marker gene next to the CMVpromoter to allow for marker selection.

For chromosomal truncation, the above TACT-constructs are transfectedinto a somatic cell hybrid line BE2CI-18-5f containing the mardel (10)chromosome. Positive selection is applied for Hygromycin or Geneticinresistance whereas negative selection is applied against the ThymidineKinase Gene. Resulting colonies are further screened with distal p′ andq′ DNA fragments to ascertain the presence or absence of the two mardel10 chromosome arms. In addition to the BE2CI-18-5f cell line, ahuman/chicken somatic cell hybrid line (derived from therecombination-proficient DT40 chicken cell line; Dieken et al., 1996)containing the mardel (10) chromosome will also be generated and used.

EXAMPLE 16 Analysis of HAC

Irrespective of which of the approaches described above is used, thepresence of a new product in a mammalian cell line as anextrachromosomal, artificial chromosome, will be assessed byfluorescence in situ hybridisation (FISH) analysis, as well as tested byextracting high molecular weight DNA to determine independently existingchromosomal entity on pulsed field gel. The stability of the constructthrough successive cell division, both in the presence and absence ofdrug-resistance selection, will be determined. The presence of theconstruct, in all or a high percentage of the original transfected cellsindicates stability. Demonstration of this stability indicates thesuccessful creation of a HAC.

EXAMPLE 17 Production of HAC

This example describes the use of the neocentromere as a source ofcentromeric DNA in the “bottom-up” approach to produce HACs in humancell culture. Bacterial artificial chromosomes (BACs) containing clonedneocentromeric DNA and a selectable marker were co-transfected withhuman telomeric DNA into human HT080 cells to yield independent HACsthat were single-copy and stable in the absence of selection. Theproperties of these HACs, and their potential utility as a new, improvedvector system for gene therapy are described.

Experimental Protocol

Preparation of DNA. Highly-purified BAC DNA was prepared using Qiagencolumns according to the manufacturer's instructions. Prior totransfection, BACs were linearized with SgrAI in the presence of 2.5 mMspermidine and examined by pulsed-field gel electrophoresis. Humantelomeric DNA was gel-purified as a 1.6-kb BamHI/BgmlII fragment frompSXneo270T2AG3 (Bianchi et al., 1997). High-molecular-weight genomic DNAwas prepared from cultured cell lines using standard methods (du Sart etal., 1997).

Transfection of RT1080 cells. Transfection of human fibrosarcoma cellline HT1080 (Rasheed et al., 1974) was performed using the DOPSERliposomal transfection reagent (Boehringer-Mannheim). The day beforetransfection, 6-well trays (each well is 962 mm²) were seeded with 3×10⁵HT1080 cells per well and grown at 37° C., 5% CO₂. Differentcombinations containing 1–2 μg of each BAC, 50 ng of telomeric DNA, 100ng of each PAC-1, 4 and 5 (du Sart et al., 1997) and 50 ng of humangenomic DNA were prepared in 50 μl of HBS (20 mM HEPES, 150 mM NaCl)supplemented with 0.075 mM spermidine and 0.030 mM spermine. These DNAcocktails were mixed with 50 μl of 0.4 μg/μl DOPSER (diluted in HBS) andleft at room temperature for 15 to 20 mm. The HT1080 cells were washedwith PBS (phosphate buffered saline) and 1 ml of serum-free DMEM(Dulbecco's modified Eagles medium) was placed in each well. TheDNA-DOPSER mixture was then added dropwise with swirling and the cellswere incubated for 6 h. 1 ml of DMEM and 20% v/v fetal calf serum (FCS)was then added and the cells left for 24 h at 37° C., 5% v/v CO₂. Thecells were harvested and seeded into 48-well cluster trays (each well is100 mm²) containing DMEM-10% v/V FCS supplemented with Geneticin (G418,Gibco-BRL) at 250 μg/ml. The media was changed every 3 to 4 days.G418-resistant colonies normally appeared 10 to 14 days aftertransfection. These colonies were expanded into duplicate 6-well trays,where the cells of one tray were stored frozen in liquid N₂, and theremaining cells were analysed by fluorescence in situ hybridization(FISH).

Cell culture and mitotic stability. HT1080 cells were grown in DMEMsupplemented with 10% v/v FCS, penicillin/streptomycin, and glutamine.The mitotic stability of HAC containing clones was determined by growthin 25 cm² flasks in the presence (200–250 μg/ml) or absence of G418selection, and grown to confluency (3–4 days) and split 1/5 and 1/10,respectively. Aliquots of each culture were harvested fortnightly andanalysed by FISH (20–50 metaphases) with BAC/E8 and/or BAC/F2 probes.

FISH, ANTI-CEN/FISH and PRINS/FISH. Fluorescence in situ hybridization(FISH) analysis of HT1080 clones was performed with BAC/E8, BAC/F2,and/or α-satellite DNA probes. Hybridization using the BAC probes wereperformed under high stringency whereas the α-satellite DNA probes wereused in low stringency conditions (du Sart et al., 1997). ANTI-CEN/FISHanalyses involved an initial immunofluorescence staining step using aCREST antibody or specific antibodies against CENP-B, CENP-C, or CENP-E,followed by FISH using the probes described above, essentially aspreviously described (du Sart et al., 1997).

Results

HAC construction strategy. The basic strategy involved theco-transfection of the 10q25.2 neocentromere DNA with human telomericDNA into human cells. The neocentromere region is cloned as two,circular YACs in Saccharomyces cerevisiae. To facilitate handling andpurification of the cloned DNA in large quantities, these YACs areretrofitted into BACs and maintained episomally in E. coli as circularmolecules. One of the BAC clones, BAC/E8, is 120 kb in size and has aninsert of 105 kb that encompassed 70 kb of the 80-kb core NC-DNA region(FIG. 16). The second BAC clone, BAC/F2, has an insert size of 75 kbthat overlapped BAC/E8 by 1.4 kb, and contains ˜10 kb of the core NC-DNAwhile extending ˜65 kb into the p′-side of the mardel (10) chromosome(FIG. 16). The BAC vector backbone further contains theneomycin-resistance (NeoR) gene to allow selection in mammalian cells.BAC/E8 and BAC/F2, used either on their own, in combination with eachother or with additional DNA are used in the following transfectionexperiments.

Transfection of RT1080 cells. The human cell line HT1080 (Rasheed etal., 1974) is chosen for the transfection experiments because of itsnear-diploid karyotype, its high level of telomerase activity (Holt etal., 1997), and its demonstrated ability to form microchromosomescontaining de novo centromeres from transfected arrays of α-satelliteDNA and human telomeric DNA (Harrington et al., 1997; Ikeno et al.,1998). The resulting G418-resistant clones are analyzed by FISH andclassified into different categories of events.

Transfected cell lines are designated HT-38, HT-47, HT-54, HT- 190, andHT-191.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variation and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individual or collectively, andany and all combinations of any two or more said steps or features.

TABLE 1 Restriction analysis of the genomic DNA of patient BE and thoseof his parents using three overlapping cosmids that span the markercentromere. Y7C14 Y6C10 Y4C7 NotI n.a. 910 910 BssHII n.a. 815, 340 n.a.BsiWI n.a. 740 740 SalI 410 410 410, 540 ClaI 315, 145, 110, 80 315,145, 110, 80 315, 145, 110, 80 SnaBI n.a. 250, 148 n.a. NaeI 240, 210,155, 120 240, 210, 155, 120 240, 210, 155, 120 NarI 222, 108, 70 222,108 222, 200, 108, 70 EclXI 180 180 180 SfiI 170 170 170 KspI 168 168168 AatII 165, 146 165, 146 165, 146 NheI 38 38 38 BstBI n.a. 35 35 SmaIn.a. 90, 40, 22 90, 40, 22 BglI 25 25, 7.2, 6.2 25 PacI n.a. 25 na.BamHI 24, 19, 15 24, 22* 24, 22* KpnI 23 23 23, 19 BclI 21 21 21 PstI9.4, 5.9, 5.1, 4.2, 3.8, 9.4, 3.8, 2.9, 2.7, 2.4, 9.4, 7.1, 4.2, 3.3,2.9, 3.3, 2.9, 2.4 1.5, 1.1 2.7, 1.9, 1.5, 1.1 XbaI 14 14, 10 10 EaeIn.a. 15, 12, 8, 6 n.a. SphI 16, 7.5 16 16, 9 PvuII 14, 7.5 7.5, 6 7.5, 6HindII 8.6, 6.9, 6.2, 2.7, 1.8, 6.9, 6.2, 5.6, 5.2, 5, 2.7, 6.2, 5.6,5.2, 4.3, 2.9, 1.2 1.9, 1.8, 1.7, 1.2, 0.6 1.7, 1.2 ApaI 15, 8.5 15 1511, 4.3, 3.9, 1.9, 1.5 11, 4, 3, 2, 1.9, 1.7, 1.5 10.2, 7.6, 3, 2, 1.9,1.7, EcoRI 1.5 HpaII 5.5, 4.3, 3.6, 1.6 6.9, 3.6, 2.8, 1.6, 1.2 3.6,2.8, 2.5, 1.6, 1.2 MspI 3.9, 3.0, 2.8, 2.5, 2, 1.6, 3.9, 3.6, 2.8, 2.5,2.2, 3.6, 3.2, 2.8, 2.5, 2.2, 1.2 1.6, 1.5, 1.3, 1.2, 0.9 1.6, 1.5, 1.2,1 SspI n.a. 10 n.a. XhoII 7.5 n.a. n.a. DraI 7.5 7.5 7.5 BglII 8.5, 6,5, 4.7, 3.5, 2.5 6, 5, 4.7, 2.5, 1.6, 1.5, 1 7, 6, 5, 4.7, 2.5, 1.6,1.5, 1.1, 1 AvaII 7.4, 3.7, 3.4, 2.8, 2.6, 3.7, 2.8, 2.6, 1.8, 1.7, 4.3,3.7, 2.8, 2.6, 1.8, 1.8, 1.7, 1.4, 1.2, 1.1 1.4, 1.2, 1.1, 0.9, 0.8,1.7, 1.4, 1.2 0.5 StuI 12.5, 8, 7.5 12.5, 9, 8.5 9, 8.5 HindIII 6.6,5.4, 4.7, 4.4, 2.9, 5, 4.7, 4.4, 4.1, 2.9, 2.5, 5, 4.7, 4.1, 3.1, 2.5,2.3, 2.5 0.7 1.9 n.a. = data not available. The values representrestriction fragment lengths in kilobases. Multiple values for an enzymedenote different bands detected by a cosmid probe on a gel lane. Sincethere were no detectable differences between the DNA of patient BE andthose of his parents in any of the fragments (except for a BamHIpolymorphic band found in one of the parents, indicated by an asterisk),only one set of values is shown for all three genomic DNA.

TABLE 2 Vectors for cloning centromeric regions from normal chromosome10 or mardel (10) DNA into yeast artificial chromosomes (YACs). TheseYACs can be shuttled into mammalian cells to test for function as HACs.Vector: Key Feature(s) Map pJS97ARTi hTEL/I-SceI/yTEL, DHFR FIG. 8ApJS98ANTi hTEL/I-SceI/yTEL, neo FIG. 8B Fragmentation 1hTEL/I-SceI/yTEL, hyg FIG. 8C Fragmentation 2 (−/+ hGH)hTEL/I-SceI/yTEL, neo, hGH FIG. 8D pVC39-AAH2 ALU-ALU TAR vector FIG. 8EpTEL/CAT/TEL hTEL/I-SceI/hTEL/neo FIG. 8F pAAH/TCNa TAR vector with FIG.8G hTEL/I-SceI/hTEL/neo pVC39-ALU/C3-F2(+/−) ALU-specifc TAR vectorsFIG. 8H pTCS ends of PAC4 in pBS FIG. 8I pTCN-TCS specific TAR vectorFIG. 8J hTEL/I-SceI/hTEL/neo

TABLE 3 PCR analysis of YAC 5f-52-E8 clone and comparison with theHC-contig/ neo-centromere region from normal chromosome 10 and mar del(10) Genomic DNA used in PCR (product size in kb) Primer-Pairs^(a)BE2C1-18-1f^(b) BE2C1-18-5f^(b) YAC 5f-52-E8 norm: 141 + 55 1.80 1.80not present norm: 32 + 30 0.90 0.90 0.90 norm: 28 + 29 1.00 1.00 1.00norm: 1 + 3 2.90 2.90 2.90 norm: 39 + 52 1.20 1.20 1.20 norm: 5 + 7 0.230.23 0.23 norm: 16 + 5 3.50 3.50 3.50 norm: 9 + 14 0.90 0.90 0.90 norm:36 + 37 2.00 2.00 2.00 norm: 168 + 71 4.00 4.00 4.00 norm: 27 + 10 15.9015.90 15.90 norm: 18 + 17 1.20 1.40 1.40 (VNTR)^(c) norm: 68 + 17 8.008.00 8.00 norm: 34 + 47 3.00 3.00 3.00 PAC4t7: a + b 0.30 0.30 notpresent AFM259xg5: ca + gt^(c) 0.21 0.19 not present ^(a)Refer to FIG.1a for the relative positions of each primer-pair. ^(b)BE2C1-18-1f andBE2C1-18-5f are somatic hybrid cell lines containing the normal humanchromosome 10 and mar del (10), respectively (2). ^(c)The ‘norm: 18 +17’ and ‘AFM259xg5: ca and gt’ primer sets allow distinction between thenormal human chromosome 10 and mar del (10) by detecting a VNTR and amicrosatellite, respectively.

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1. An isolated nucleic acid molecule comprising a neocentromere, whereinsaid neocentromere comprises a region of an eukaryotic chromosome anddoes not have any detectable alpha satellite DNA as determined byfluorescent in situ hybridisation (FISH), wherein said nucleic acidmolecule comprises SEQ ID NO: 3, and wherein said nucleic acid molecule,when introduced into a cell, is capable of replicating, acting as anextra-chromosomal element and segregating with cell division.
 2. Theisolated nucleic acid molecule according to claim 1 wherein theeukaryotic chromosome is a mammalian chromosome.
 3. The isolated nucleicacid molecule according to claim 2 wherein the chromosome is a humanchromosome.
 4. The isolated nucleic acid molecule according to claim 2wherein the nucleic acid molecule binds to centromeric binding proteins(CENP)-A and -C or antibodies thereto.
 5. The isolated nucleic acidmolecule according to claim 3 wherein the chromosome is human chromosome10.
 6. The isolated nucleic acid molecule according to claim 5 whereinsaid neocentromere comprises a region mapping between q24 and q26 onsaid human chromosome
 10. 7. The isolated nucleic acid moleculeaccording to claim 3 wherein said human chromosome is a mardel (10)chromosome.
 8. The isolated nucleic acid molecule of claim 1 whereinsaid nucleic acid molecule is in linear form and co-introduced into acell together with a telomeric sequence.
 9. The isolated nucleic acidmolecule according to claim 8 wherein the eukaryotic chromosome is amammalian chromosome.
 10. The isolated nucleic acid molecule accordingto claim 9 wherein said nucleic acid molecule binds to CENP-A and CENP-Cantibodies.
 11. The isolated nucleic acid molecule according to claim 9wherein the mammalian chromosome is human chromosome
 10. 12. Theisolated nucleic acid molecule according to claim 11 wherein theneocentromere comprises a region mapping between q24 and q26 on saidhuman chromosome
 10. 13. The isolated nucleic acid molecule according toclaim 8 wherein said chromosome is a human mardel (10) chromosome.
 14. Agenetic construct comprising an origin of replication for a eukaryoticcell and the nucleic acid molecule of claim 1, operably linked totelomeric nucleotide sequences functional in the cell in which thegenetic construct is to replicate and wherein said genetic constructswhen introduced into a cell, is a replicating, extra-chromosomal elementwhich segregates with cell division.
 15. The genetic construct accordingto claim 14 wherein the eukaryotic chromosome is a mammalian chromosome.16. The genetic construct according to claim 15 wherein the eukaryoticchromosome is a human chromosome.
 17. The genetic construct according toclaim 16 wherein the nucleic acid molecule binds to CENP-A and -C orantibodies thereto.
 18. The genetic construct according to claim 17wherein the neocentromere is from human chromosome
 10. 19. The geneticconstruct according to claim 18 wherein the neocentromere comprises aregion between q24 and q26 on said human chromosome
 10. 20. The geneticconstruct according to claim 18 wherein said chromosome is a humanmardel (10) chromosome.